The 2Fe2S centres of the 2-oxo-1,2-dihydroquinoline 8-monooxygenase from Pseudomonas putida 86 studied by EPR spectroscopy

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ELSEVIER

Biochi~ic~a et BiophysicaA~ta Biochimica et Biophysica Acta 1252 (1995) 177-179

Rapid Report

The 2Fe2S centres of the 2-oxo-l,2-dihydroquinoline 8-monooxygenase from Pseudomonas putida 86 studied by EPR spectroscopy Bettina Rosche a, Susanne Fetzner a, Franz Lingens a, Wolfgang Nitschke b, Astrid Riedel c,, a lnstitutfiir Mikrobiologie, Universitiit Hohenheim, D-70593 Stuttgart, Germany b lnstitutfiir Botanik 11, Universiti~t Freiburg, D-79104 Freiburg, Germany c lnstitutf~i r Biophysik und Physikalische Biochemie, Universitiit Regensburg, D-93040 Regensburg, Germany Received 5 June 1995; accepted 7 July 1995

Abstract

The 2-oxo-l,2-dihydroquinoline 8-monooxygenase from Pseudomonas putida 86 comprises two components with four redox active sites necessary for activity. We present an EPR characterization of the iron-sulfur centres in the purified reductase and oxygenase component of this novel enzyme system. The oxygenase component was identified as a Rieske [2Fe2S] protein on the basis of its characteristic EPR spectrum with gz,y,x = 2.01, 1.91, 1.76 and gay = 1.893. The reductase component, an iron-sulfur flavoprotein, contained a [2Fe2S] cluster with gz.y.x = 2.03, 1.94, 1.89 and the average g-value (gay) of 1.953, typical of a ferredoxin-type centre. In redox titrations at pH 7, the midpoint potentials were determined to be - 180 mV + 30 mV and - 100 mV + 10 mV for the reductase and oxygenase component, respectively. A detailed comparison to other muiticomponent enzyme systems is presented pointing out the EPR and redox properties of the FeS centres involved. Keywords: EPR; Iron-sulfur centre; Rieske centre; Ferredoxin; Monooxygenase; Dioxygenase; (P. putida)

Pseudomonas putida 86 has been shown to utilize quinoline as sole source of carbon, nitrogen and energy [1]. The second step of this catabolic pathway is catalyzed by 2-oxo-l,2-dihydroquinoline 8-monooxygenase, the function of which is dependent upon NADH and Fe 2÷. It consists of a 2Fe2S-flavo]?rotein as reductase and an oxygenase component with a further 2Fe2S-cluster and additional iron. The purification procedure as well as the optical and kinetic measurements are published in [2]. Here, we present the EPR results (spectra, g-values, redox midpoint potentials) obtained for the 2Fe2S centres of the 2-oxo- 1,2-dihydroquinoline 8-monooxygenase. A multitude of non-heine iron (mono- and di-) oxygenases has been characteriized and classified according to the number of protein components, the type of flavins and iron-sulfur centres invoNed in the electron transport from

Abbreviations: Era, redox midpoint potential; Fd, ferredoxin-type cluster; gay, average g-value =: 1 / 3 ( g z + gy + gx); Mops, 3'-(N-morpholino)propanesulfonic acid. * Corresponding author. Tel.: +49 941 9432185; fax: +49 941 9432479; e-mail: [email protected]. 0167-4838/95//$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0 1 6 7 - 4 8 3 8 ( 9 5 ) 0 0 1 5 1 - 4

NAD(P)H to molecular oxygen [3,4]. However, up to now, no systematical comparison is available summarizing the g-factor values and midpoint potentials of the FeS centres. They are usually divided into two groups, i.e. the ferredoxin-type and the Rieske-type clusters. This classification is based on the average g-factor derived from the EPR spectrum. In addition to the characterization of the ironsulfur centres in the 2-oxo-l,2-dihydroquinoline 8-monooxygenase, we give a survey of previously published EPR data taking into account the number of protein components, their subunit structure, the redox centres and midpoint potentials using the nomenclature given in [3]. The purification and biochemical characterization of the enzyme components are published in [2]. Redox titrations were carried out as described in [5], at pH 7 in the presence of 50 mM Mops. The pH values were controlled at the beginning and the end of each titration. The following redox mediators were used at 50 p,M (a) for the titration of the ferredoxin-type cluster: Neutral red ( - 325 mV), Safranine T ( - 2 8 9 mV), anthraquinone-2-sulfonate ( - 2 2 5 mV), anthraquinone-2-6-disulfonate ( - 1 8 4 mV), 2-hydroxy-l,4-naphthoquinone ( - 1 4 5 mV), 5-dihydroxyp-benzoquinone ( - 6 0 mV), tetramethyl-p-benzoquinone

B. Rosche et al./ Biochimica et Biophysica Acta 1252 (1995) 177-179

178

( + 5 mV), 1,4-naphthoquinone ( + 60 mV), 1,2-naphthoquinone ( + 145 mV), and (b) for the titration of the Rieske-type centre: Neutral red ( - 3 2 5 mV), Safranine T ( - 2 8 9 mV), anthraquinone-2-6-disulfonate ( - 1 8 4 mV), anthraquinone-1,5-disulfonate ( - 170 mV), Indigo carmine ( - 1 2 5 mV), Methylene blue ( + 11 mV), 5-hydroxy-l,4naphthoquinone ( + 30 mV), phenazine ethosulfate ( + 55 mV), Toluylene blue ( + I 15 mV). For the reductive titrations sodium dithionite was used, the oxidative titrations were done using ferricyanide. The EPR spectra were taken on a X-band Bruker spectrometer fitted with an Oxford Helium cryostat and temperature control system. Fig. 1 shows the EPR spectra of the 2Fe2S centres in the 2-oxo-l,2-dihydroquinoline 8-monooxygenase from Pseudomonas putida 86 following reduction with sodium dithionite (50 mM) at pH 7. The oxygenase component exhibits a Rieske-type spectrum with g~ = 2.01, g,, = 1.91 and gx = 1.76 and a gay value of 1.893 (Fig. la). The spectrum of the reductase component shows signals at gz = 2.03, gy = 1.94 and gx = 1.89 and is significant for a ferredoxin-type iron-sulfur cluster with its lower g-factor anisotropy resulting in a higher gav of 1.953 (Fig. lb). The flavosemiquinone radical identified as FAD in [2]

i 2,0-~

i

i

i

aflfl ~1,91

~1,89

MagnetiFiecl[roT] d 38O 3;,0

3;0

'

Fig. 1. EPR spectra and g-values of the 2Fe2S centres of the 2-oxo-l,2dihydroquinoline 8-monooxygenase from Pseudomonas putida 86. The spectra were taken on the purified enzyme components (a, oxygenase; b, reductase) in the presence of 50 mM sodium dithionite. Instrument settings: microwave frequency, 9.47 GHz; modulation amplitude, 1.6 mT; microwave power, 6.3 mW; temperature, 15 K.

Table 1 EPR and redox properties of some multicomponent non-heme iron oxygenases [Ref]

Protein/integral redox sites/ subunit composition

2Fe2S

Class IA: Phthalate dioxygenase [8] reductase/FMN, 2Fe2S Fd [10] oxygenase/2Fe2S/ol 4 Rieske Class IA: 4-Methoxybenzoate monooxygenase [6] reductase/FMN, 2Fe2S Fd [6] oxygenase/2Fe2S/a 3 or oq Rieske Class IA: 4-Chlorophenylacetate 3,4-dioxygenase [ 13] reductase/FMN, 2Fe2S Fd [ 15] oxygenase/2Fe2S/ot 3 Rieske Class IB: 2-Halobenzoate 1,2-dioxygenase [ 16] reductase/FAD, 2Fe2S Fd [16] oxygenase/2Fe2S/°~3 f13 Rieske Class IB: 2-Oxo- l,2-dihydroquinoline 8-monooxygenase [2] reductase/FAD, 2Fe2S Fd [2] oxygenase/2Fe2S/a 6 Rieske Class IlA: Pyrazon dioxygenase [ 17] reductase/FAD [17] iron-sulfur-protein/2Fe2S Fd [17] oxygenase/2Fe2S Rieske Class liB: Benzene dioxvgenase [ 18] reductase/FAD [ 18] iron-sulfur protein/2Fe2S Rieske [18] °xygenase/2Fe2S/a2 ~2 Rieske Class liB: Toluene dioxygenase [ 19] reductase/FAD [20] iron-sulfur protein/2Fe2S Rieske [21 ] °xygenase/2Fe2S/°~2 f12 Class 111: Naphthalene dioxygenase [22] reductase/FAD, 2Fe2S Fd [23] iron-sulfur protein/2Fe2S Rieske [24] °xygenase/2Fe2S/°z2 f12 Rieske

g:

gy

gx

ga,.

E m [Ref]

2.041 2.016

1.949 1.914

1.900 1.763

1.963 1.898

- 174 m V / p H 7 [9] - 6 0 m V / p H 6.9 [111 ~

2.023 2.008

1.942 1.913

1.893 1.72

1.953 1.880

0 m V / p H 7.8 [12]

2.043 2.021

1.951 1.922

1.891 1.737

1.962 1.893

- 171 m V / p H 7 [14] - 6 2 m V / p H 7 [14]

2.043 2.025

1.951 1.912

1.891 1.788

1.962 1.908

- 200 m V / p H 7 b -- 125 m V / p H 7 b

2.03 2.01

1.94 1.91

1.89 1.76

1.953 1.893

-- 180 m V / p H 7.1 - 100 m V / p H 7.2

2.02 2.02

1.94 1.91

1.79

1.907

2.026 2.018

1.890 1.917

1.834 1.754

1.917 1.896

- 155 m V / p H 7 [18] - 112 m V / p H 7 [18]

2.01

1.86

1.81

1.893

-109mY[20]

2.01

1.91

1.80

1.907

a In [9], however, an E m of - 120 mV at pH 7.0 is given for the FeS-centre of the oxygenase. b Riedel, A., and Fetzner, S., unpublished results.

B. Rosche et al./Biochimica et Biophysica Acta 1252 (1995) 177-179

and usually seen as a g = 2.003 signal in EPR was not observed in the spectrum, since the flavin was most probably in the fully reduced state after addition of dithionite. The midpoint potentials were determined by redox titrations monitoring the dependence of the EPR signals on ambient potential. The data points could be fitted to a theoretical Nernst curve 'with n = 1. The midpoint potential of the reductase was determined to be - 180 mV at pH 7.1 with an experimental error of + 3 0 mV due the instability of the enzyme during the titration. The E m value of the oxygenase was found to be - 100 mV _ 10 mV at pH 7.2. The 2-oxo-1,2-dihydroquinoline 8-monooxygenase from Pseudomonas putida 86 is an example of a class IB enzyme system [2] according to the classification of [3]. It comprises two components with four redox active centres, FAD, a ferredoxin- and a Rieske-type 2Fe2S-cluster and probably mononuclear Fe 2+. (It is of note, however, that, among all multicomponent enzyme systems, the involvement of Fe 2÷ is only proven for the 4-methoxybenzoate monooxygenase [6].) The reductase part containing both FAD and a ferredoxin
179

criterion does not hold any longer. In the region between - 2 0 0 mV and - 1 0 0 mV, both ferredoxin- and Riesketype centres are found.

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